The entire text of the above-referenced disclosure is specifically incorporated by reference herein without disclaimer.
1. Field of the Invention
The present invention relates generally to the field of molecular biology, and more particularly, DNA repair. In specific embodiments, the invention relates to DNA sequences encoding fusions of DNA repair proteins having complementary functions, and to the use of such DNA sequences to enhance the survival of cells when subjected to DNA-damaging agents such as chemotherapeutics.
2. Description of Related Art
The use of multiagent chemotherapy protocols has produced dramatic increases in the survival rates of many cancers. Pediatric cancers have been particularly amenable to treatments utilizing multiagent and multimodality approaches. In addition, dose intensification has been increasingly utilized in attempts to increase survival rates of both adult and pediatric cancers. DNA alkylating agents have been an important part of most dose-intensification protocols.
The positive impact of increased dose intensity of therapy on response rate and survival duration has been demonstrated in childhood Burkitt's lymphoma, metastatic breast cancer, neuroblastoma, testicular cancer and osteogenic and Ewing's sarcoma (Broun et al., 1992; Schwenn et al., 1991; Cheung and Heller, 1991; Smith et al., 1991) and most recently in childhood acute myelocytic leukemia (Woods et al., 1996). Invariably, and in spite of the increasing use of myeloid growth factor support, myelosuppression is a major impediment to further dose-intensification in humans. This is particularly true in patients with relapsed disease previously treated with intensive chemotherapy as initial therapy.
One approach to circumvent the dose-limiting myeloid toxicities of chemotherapy agents has been the use of recombinant vectors to introduce and express various genes important in chemotherapy resistance in bone marrow-derived cells (Moritz and Williams, 1996). The majority of work in this area has been focused on the use of recombinant retrovirus vectors. Gene transfer has been accomplished using several different DNA sequences, including dihydrofolate reductase (DHFR) or DHFR mutants encoding resistance to methotrexate (Williams et al., 1987; Miller et al., 1984; Li et al., 1994; Corey et al., 1990), p-glycoprotein multidrug resistance (MDR-1) gene encoding resistance to several alkylating agents (Hanania and Deisseroth, 1994), glutathione-dependent enzymes encoding resistance to alkylating agents and ionizing radiation (Greenbaum et al., 1994) and cytosolic aldehyde dehydrogenase encoding resistance to cyclophosphamide (Magni et al., 1996). More recently, work has been concentrated on retroviral vectors encoding the DNA repair protein 0-6-methylguanine-DNA methyltransferase (MGMT) as a mechanism to generate resistance to chloroethylnitrosourea (CENUs) and other alkylating agents (Moritz et al., 1993; Moritz et al., 1995; Maze et al., 1996).
Chloro-ethyl-nitrosoureas (CENUs) have been shown to be effective agents in treatment of several human cancers, particularly brain tumors. A major determinant of CENU-induced cytotoxicity is the alkylation of guanine at the O.sup.6 -position and the formation of interstrand DNA crosslinks. While alkylation at the O.sup.6 -position primarily induces G:C to A:T transition, interstrand DNA crosslinks are particularly cytotoxic because they disrupt DNA replication (Toorchen and Topel, 1983). CENU-induced DNA adducts, such as a chloroethyl group at the O.sup.6 -position, can initiate the subsequent formation of an interstrand crosslink by rearranging to produce an ethyl bridge between N1 of guanine and N3 of cytosine in the opposite strand (Ludlum, 1980). Repair of this lesion is distinct, since it involves direct reversal of the damaged adduct by the mammalian protein MGMT (Erickson et al., 1980; Robins et al., 1983; Samson et al., 1986; Brent and Remack, 1988).
MGMT transfers the chloroethyl group from guanine to an internal cysteine residue located within the acceptor site of the MGMT protein and thus repairs the modified base prior to the formation of the interstrand crosslink (Saffhill et al., 1985; Pegg et al., 1995). In most cases, the level of MGMT protein in mammalian cells correlates with CENU sensitivity (Erickson et al., 1980; Pegg et al., 1995; Lindahl et al., 1988). The amount of MGMT protein expressed in human and murine bone marrow cells is considerably lower than in other tissues and contributes to the inefficient repair of CENU-induced DNA damage in blood cells (Moritz et al., 1995; Gerson et al., 1985). Thus increased expression of MGMT via gene transfer provides a unique opportunity to effect drug resistance by increasing the expression of an endogenous protein.
Several laboratories have demonstrated that transduction of murine or human hematopoietic stem and/or progenitor cells, via a retroviral vector encoding the human MGMT cDNA, protects bone marrow cells from CENU-induced myelotoxicity (Moritz et al., 1995; Maze et al., 1996; Allay et al., 1995; Jelinek et al., 1996; Wang et al., 1996). In previous studies, a model of CENU-induced fatal bone marrow suppression was developed (Maze et al., 1994). Reconstitution of murine bone marrow with hematopoietic stem cells expressing vector-derived MGMT protected mice from 1,3, Bis (2-chloroethyl)-nitrosurea (BCNU)-induced bone marrow hypoplasia and peripheral blood pancytopenia (Moritz et al., 1995; Maze et al., 1996). Bone marrow cells harvested from these mice were more resistant to BCNU in vitro and demonstrated a higher level of MGMT DNA repair activity compared to BCNU-treated mock-infected control mice (Maze et al., 1996). In addition, a significant reduction in short-term CENU-related mortality was observed in BCNU-treated mice transplanted with MGMT-expressing hematopoietic stem cells (Maze et al., 1996).
In light of the foregoing discussion, it is clear that there is a limitation with current chemotherapies in that the ability to increase doses of chemotherapy used to treat cancer patients is inhibited by the cytotoxicity of the chemotherapeutic agents on various non-target organ systems including the bone marrow. If DNA repair genes that protect against the deleterious effects of chemotherapeutic agents with oxidative damaging capacity can be inserted into the patient's bone marrow cells, or other organ systems prone to damage by these agents (such as lung), these systems can be protected and possibly the dose of treatment increased to rid the system of the cancer. Furthermore, although this idea has been tested with the MGMT gene, this gene is only limited to repairing the O.sup.6 guanine lesion that occurs from various chemotherapeutic agents, especially the chloronitrosoureas. However, a large number of chemotherapeutic agents also cause damage at other nucleophilic sites in the DNA, such as N.sup.7 -guanine, N.sup.3 -adenine, etc. Furthermore, a number of these agents also are oxidative DNA damaging agents and can have deleterious effects on the patient via this pathway. In light of the foregoing, it is evident that there remains a need for improved methods for enhancing the protection of non-target cells when prone to or subjected to DNA-damaging agents, such as chemotherapeutic agents or in malignant conditions such as Fanconi's anemia. The present invention is addressed to these needs.